One reason why pancreatic cancer is so aggressive and unresponsive to treatments is its resistance to apoptosis. Growth factors both stimulate proliferation and protect pancreatic cancer (PaCa) cells from death. In particular, insulin-like growth factor I (IGF-I) plays a major role in PaCa cell survival. We have found that reactive oxygen species (ROS) produced by NADPH oxidase mediate the pro-survival effect of IGF-I in these cells. The overall goal of the proposed project is to determine the mechanisms of the pro-survival effect of NADPH oxidase in PaCa cells. We will determine the mechanism of NADPH oxidase activation in PaCa cells, which remains an unresolved issue of general importance for non- phagocytic cells. In particular, we plan to determine the involvement of different subunits, such as Nox4 and p22, and the role of transcriptional regulation of subunits'expression in the IGF-I induced activation of NADPH oxidase. We hypothesize that ROS generated by NADPH oxidase enhance and prolong the activation of PI3K/Akt, a major anti-apoptotic pathway. This pro-survival pathway is tightly regulated by kinases and protein tyrosine phosphatases (PTPs). Although the role of kinases has been extensively studied, much less is known on the involvement of PTPs in the anti-apoptotic effects of growth factors. PTPs are highly sensitive to and undergo reversible inhibition by ROS. We further hypothesize that ROS produced by NADPH oxidase inhibit specific PTPs, such as LMW-PTP and PTP1B, which negatively regulate the PISK/Akt pathway. The inhibition of these PTPs by ROS is necessary for growth factors, in particular IGF-I, to enhance and maintain the activation of PI3K/Akt. An understanding of the protective, anti-apoptotic role of NADPH oxidase in PaCa cell death will allow us to propose novel therapeutic strategies for pancreatic cancer. In particular, inhibition of NADPH oxidase or related signaling pathways could be one strategy to stimulate apoptosis and thus overcome pancreatic cancer resistance to chemo- and radiation therapies. The specific objectives of our proposal are (1) determine the role of NADPH oxidase subunits in its activation by IGF-I in PaCa cells;(2) determine the role of subunits'transcriptional up-regulation in NADPH oxidase activation induced by IGF-I in PaCa cells;(3) determine the effect of IGF-I on LMW-PTP and PTP1B, and the regulation of these PTPs by NADPH oxidase in PaCa cells;(4) determine the roles of LMW-PTP and PTP1B in PISK/Akt activation and inhibition of apoptosis by IGF-I in PaCa cells;(5) determine the effects of NADPH oxidase inhibition on tumorigenesis in an in vivo model of pancreatic cancer.